The present invention relates to a nucleic acid construct for expressing active substances which can be activated by proteases and to its preparation and use.
Like inflamed areas, tumors are distinguished from the surrounding normal tissue by a substantial increase in the formation and secretion of proteases [Schmitt et al., Fibrinol. 6, 3 (1992), Cottam et al., Int. J. Oncol. 2, 861 (1993), Tryggvason et al., Breast Cancer Res. And Treatm. 24, 209 (1993), Leto et al., Anticancer Res. 12, 235 (1992), Hart, Fibrinol. 6, 11 (1992), Albini et al., J. Natl. Cancer Inst. 83, 735 (1991)]. Examples of these proteases are plasminogen activators, catnepsins and matrix metalloproteinases.
An essential function of these tumor proteases is to dissolve the extracellular matrix to allow the tumor cells to invade, and grow in an infiltrative manner in, normal tissue. At the same time, these proteases protect the tumor from the defence mechanisms of the body insofar as the active compounds which are required for defence are cleaved, and thereby inactivated, by the proteases which are formed by the tumor. Thus, for example, antibodies, cytokines and growth factors, complement factors, coagulation factors and mediators are inactivated by tumor proteases.
In the past, the aim was, therefore, to inhibit the infiltrative growth and metastatic growth of tumors, and inactivation of the defence mechanisms of the body, by inhibiting the tumor cell proteases [Hocman, Int. J. Biochem. 24, 1365 (1992), Troll et al., JNCI 73, 1245 (1984), Ray et al., Eur. Respir. 7, 2062 (1994), Koop et al., Cancer Res. 54, 4791 (1994), Chiriri et al., Int. J. Cancer 58, 460 (1994), Denhardt et al., 59, 329 (1993), Melchiori et al., Cancer Res. 52, 2353 (1992)]. However, particularly for stoichiometric and pharmacokinetic reasons, little success has previously been achieved in inhibiting tumor cell proteases.
An attempt was therefore made to use the tumor cell proteases to activate bacterial toxins such as Staphylococcus aureus xcex1-hemolysin [Panchal et al., Nature Biotechn. 14, 852 (1996)). For this, an amino acid sequence (SEQ ID NO:1), i.e. XX-Arg-X, was inserted into positions 129 to 132 of the xcex1-hemolysin and in this way inactive mutants were produced which are only cleaved, and thereby activated, by tumor proteases such as cathepsin B.
Based on these results, proimmunolysins were proposed [Panchal et al., Nature Biotechn. 14, 852 (1996)], which proimmunolysins comprise an antibody which is coupled to a Staphylococcus, aureus xcex1-hemolysin which can be activated by tumor proteases or to a sea anemone equinatoxin II, with the antibody determining the target cell specificity of the coupling product.
However, the proposed concept suffers from the following disadvantages in relation to its use in tumor therapy:
In the first place, the authors chose xenogeneic nonendogenous lysins and/or toxins which are immunogenic for the host organism (for example, patients) and as a result induce an immune reaction in the host organism, which immune reaction neutralizes and inactivates the antibody/toxin conjugate. In the second place, it is known [Sedlacek et al., Antibodies as Carriers of Cytotoxicity, Contrib. to Oncol. 43, Karger Verlag, Munich, 1992] that, due to their molecular size and to the Theological conditions at the tumor, tumor-specific antibodies and immunotoxins only accrue in very small quantities (0.01-0.001% of the given antibody or immunotoxin/g of tumor) at the tumor and only penetrate the tumor to an incomplete extent so that it is either not possible to destroy all the tumor cells or only possible to destroy a small portion of the cells of a tumor. Then again, the extent to which tumor antigens, against which the antibody is directed, are expressed usually differs between the individual tumor cells, and the variable, antigen-negative tumor cells readily evade the attack by the antibodies or the immunotoxins. In addition to this, antigens which are secreted by the tumor cells neutralize the antibodies at the periphery of the tumor (Sedlacek et al., Monoclonal Antibodies in Tumor Therapy, Contrib. to Oncol., Karger Verlag, 1988).
Consequently, there is still a great need for a target cell-specific therapy for tumors and inflammations.
An object of the present invention is therefore to provide an active compound against tumors and inflammations, which active compound does not exhibit said disadvantages. The present invention therefore relates to a novel technique which uses the secretion of enzymes in tumors or areas of inflammation to achieve the local release of active compounds whose inactive precursors are expressed in tumor cells, tumor-associated cells or inflammatory cells.
One part of the subject-matter of the present invention is therefore a nucleic acid construct for expressing an active substance which is activated by an enzyme which is released from mammalian cells, which nucleic acid construct comprises the following components:
a) at least one promoter element,
b) at least one DNA sequence which encodes an active compound (protein B),
c) at least one DNA sequence which encodes an amino acid sequence (part structure C) which can be cleaved specifically by an enzyme which is released from a mammalian cell, and
d) at least one DNA sequence which encodes a peptide or protein (part structure D) which is bound to the active compound (protein B) by way of the cleavable amino acid sequence (part structure C) and inhibits the activity of the active compound (protein B).
Further objects of the instant invention are described as follows:
1. A nucleic acid construct for expressing an active substance which is activated by an enzyme which is released from mammalian cells, wherein the construct comprises the following nucleic acid sequences in the following order:
a) at least one promoter element operably linked to;
b) at least one nucleic acid sequence which encodes an active compound, wherein the active compound is endogenous to mammals, operably linked to;
c) at least one nucleic acid sequence which encodes an amino acid sequence cleavable specifically by an enzyme which is released from a mammalian cell, operably linked to;
d) at least one DNA sequence which encodes a polypeptide which is bound to the active compound by the cleavable amino acid sequence and inhibits the activity of the active compound, and wherein the nucleic acid component c) does not naturally occur as operably linking the nucleic acid sequence b) to the nucleic acid d).
2. A nucleic acid construct as described in 1, wherein the enzyme is a protease.
3. A nucleic acid construct as described in 1, wherein the enzyme is a prostate specific antigen, a plasminogen activator, a cathepsin or a matrix metalloproteinase.
4. A nucleic acid construct as described in 1, wherein the mammalian cells are tumor cells, leukemia cells, endothelial cells, macrophages, lymphocytes, muscle cells, epithelial cells, glia cells, synovial cells or virus-infected cells.
5. A nucleic acid construct as described in 1, wherein the nucleic acid construct further comprises a nucleic acid sequence operably linked to the construct of 1, wherein the nucleic acid sequence encodes a ligand which binds the active compound to a target structure.
6. A nucleic acid construct as described in 1, wherein the nucleic acid sequences b) and d) of 1 encode parts of a natural precursor of a protein active compound, wherein the nucleic acid sequence encoding the, cleavage sequence naturally occurring between the nucleic acid sequences b) and d) has been replaced by the nucleic acid sequence c), which does not naturally occur between the nucleic acid sequences b) and d).
7. A nucleic acid construct as described in 1, wherein the polypeptide encoded by the nucleic acid sequence d) is part of a natural precursor of a protein active compound.
8. A nucleic acid construct as described in 1, wherein the construct is operably inserted into a plasmid or a viral vector.
9. A nucleic acid construct as described in 1, wherein the nucleic acid sequence a) is a promoter sequence which can be activated nonspecifically, cell-specifically, virus-specifically, metabolically, cell cycle-specifically or by tetracycline.
10. A nucleic acid construct as described in 1, wherein the nucleic acid sequence a) comprises at least two identical or two different promoter sequences.
11. A nucleic acid construct as described 9, wherein the nucleic acid sequence a) is activated in endothelial cells, in cells adjoining activated endothelial cells, in muscle cells, in leukemia cells, in tumor cells, in glia cells, in lymphocytes, in macrophages or in synovial cells.
12. A nucleic acid construct as described in 1, wherein the active compound activates or inhibits a biological activation cascade or is an active component of this cascade, or activates or inhibits the coagulation system, activates fibrinolysis, activates the complement system or activates the kinin system, or is an enzyme which converts the inactive precursor of a pharmacological substance into the active substance, or which itself is a pharmacologically active substance.
13. A nucleic acid construct as described in 12, wherein the active compound is a coagulation factor which is selected from the group consisting of thrombin, factor Va, factor VIIa, factor IXa, factor Xa, TF coagulation-active fragments or factor XIIa; thrombin which is mutated in the region of the Arg-Thr cleavage site (amino acid position 327/328); a fibrinolytic protein which is selected from urokinase, tPA or functional hybrids thereof; a complement factor which is selected from CVF, C3b or functional cleavage products thereof; an antithrombotic protein which is selected from protein C, C-1S inhibitor, al-antitrypsin, hirudin, AT-III, TFPI, PAI-1, PAI-2 or PAI-3; a kallikrein; a cytostatic, cytotoxic or inflammation-eliciting protein; an antiangiogenic protein; an immunomodulatory protein; an antiinflammatory protein; a protein which relieves damage to the nervous system; a protein which inhibits or neutralizes the neurotoxic effect of TNFxcex1; an angiogenesis-stimulating protein; a hypotensive protein; an antiviral protein; a cytokine; an interferon; a tumor necrosis factor; oncostatin M or LIF; a cytokine receptor; the moiety of a cytokine receptor which is external to the cell; a cytokine antagonist; a growth factor; a growth factor receptor; the moiety of a growth factor receptor which is external to the cell; a chemokine; angiostatin; platelet factor 4; TIMP-1, TIMP-2 or TIMP-3; a nitroreductase; a xcex2-glucuronidase; a carboxypeptidase; a xcex2-lactamase; a cytosine deaminase; a catalase; a peroxidase; a phosphatase; an oxidase; kallikrein or an endothelial cell nitric oxide synthase.
14. A nucleic acid construct as described in 1, which further comprises a nucleic acid sequence bxe2x80x2) which encodes a ligand which binds to a cell membrane receptor, a cell membrane antigen, a cell membrane-located adhesion molecule, or to the extracellular matrix or component thereof.
15. A nucleic acid construct as described in 14, wherein the ligand is an antibody or an antibody fragment which binds specifically to a cell membrane antigen or to an antigen on the extracellular matrix, or is a polypeptide which binds to receptor on the cell membrane wherein the polypeptide is a growth factor, a cytokine, an interferon, a tumor necrosis factor, a chemokine, a receptor-binding part sequence of these ligands, a peptide hormone, angiotensin, kinin, folic acid, an adhesion molecule or the part sequence of the adhesion molecule which binds to the corresponding adhesion molecule or to the extracellular matrix, an extracellular moiety of an Fc receptor, a glycoprotein of a virus, a part sequence of the glycoprotein which binds to these cells, the transmembrane domain of a receptor or of a viral glycoprotein, or a glycophospholipid anchor.
16. A nucleic acid construct as described in 14, wherein the ligand binds to activated or proliferating endothelial cells, to tumor cells, to muscle cells, preferably smooth muscle cells, to fibroblasts, to macrophages, to lymphocytes, to liver cells, to kidney cells, to synovial cells, to inflammatory cells, to virus-infected cells, to bronchial epithelial cells, to glia cells or to leukemia cells.
17. A nucleic acid construct as described in 14, wherein the construct comprises at least two identical or different nucleic acid sequences b)c)d) or bxe2x80x2)b)c)d), which nucleic acid sequences are linked to each other by way of an internal ribosomal entry site.
18. A process for preparing a nucleic acid construct according to 1, which comprises operably linking the nucleic acid sequences of 1.
19. A method for the treatment or prophylaxis of tumors, leukemias, allergies, autoimmune diseases, infections, inflammations, transplant rejection reactions, thromboses, blood vessel occlusions, blood coagulation, blood circulation disturbances, injuries to tissues, or damage to the nervous system, comprising administering to a mammal an effective amount of a polypeptide expressed by the nucleic acid construct of 1.
20. A method for preparing a recombinantly altered cell, comprising transducing a suitable cell with the nucleic acid construct of 1.
21. A method for preparing a polypeptide which is encoded by the nucleic acid construct of 1, comprising transducing a suitable cell with the construct, expressing the polypeptide in the cell, and isolating the expressed polypeptide.
22. The method of 20, wherein the cell is an endothelial cell, a lymphocyte, a macrophage, a glia cell, a fibroblast, a liver cell, a kidney cell, a muscle cell, a cell of the bone or cartilage tissue, a synovial cell, a peritoneal cell, a skin cell, an epithelial cell, a leukemia cell or a tumor cell.
23. The method of 21, wherein the cell is an endothelial cell, a lymphocyte, a macrophage, a glia cell, a fibroblast, a liver cell, a kidney cell, a muscle cell, a cell of the bone or cartilage tissue, a synovial cell, a peritoneal cell, a skin cell, an epithelial cell, a leukemia cell or a tumor cell.
24. A cell transduced with the nucleic acid construct of 1.
25. A protein encoded by the nucleic acid construct of 1.
The term xe2x80x9cendogenous to mammalsxe2x80x9d as used to describe the active compound of the instant invention denotes a polypeptide that is naturally expressed in mammals or a derivative thereof as discussed herein.
The term xe2x80x9cdoes not naturally occurxe2x80x9d as used to describe the linking nucleic acid component c) denotes that the described component c) of the instant invention is not found in nature as operably linking components b) and d).